1. Technical Field
The present invention relates to a DNA encoding a novel fusion protein which is used for preparing recombinant insulin. More specifically, the present invention relates to the use of the DNA for the preparation of insulin from the fusion protein, which is obtained by the expression of the DNA, through the action of thrombin and carboxypeptidase B.
2. Background Art
Insulin is a hormone secreted by the B cells of the islet of Langerhans in the pancreas when an animal ingests food, which is the most important hormone for storage or use of sugars, amino acids and fatty acids and for maintaining the blood sugar homeostasis. Although blood sugar, namely glucose in blood, is an essential energy source for a living body, if the blood sugar homeostasis is not maintained, then serious conditions may develop. Increased blood sugar level causes the excretion of sugar in the urine, resulting in loss of glucose, i.e. onset of a so-called diabetes. If this condition continues for long periods, complications can develop in the tissues of a living body. On the other hand, decreased blood sugar level leads to an insufficient supply of the energy source, resulting in imperilment of life. Homeostasis of the blood sugar level is maintained by balancing factors that act to increase the blood sugar level (e.g., glucagon, growth hormone, cortisol, catecholamine) with factors that act to decrease the blood sugar level. Insulin is the only hormone which can decrease the blood sugar level. Hence, the reduction in secretion functions resulted from some causes and, as a consequence, insufficient supply of insulin can induce insulin-dependent diabetes mellitus (IDDM). For patients suffering from such a disease, insulin is an indispensable drug.
Human insulin is a polypeptide comprising an A chain with 21 amino acids and a B chain with 30 amino acids, which has one intrachain disulfide bond in the A chain and two disulfide bonds which link between the A chain and the B chain. Insulin is initially biosynthesized as “preproinsulin” on ribosomes in B cells of the islets of Langerhans of the pancreas. Preproinsulin is a linear molecule comprising a signal peptide with 24 amino acids (SP), a B chain (B), a C-peptide with 31 amino acids (C) and an A chain (A) linked in the order as represented by the formula “SP-B-C-A”. Upon transport to the endoplasmic reticulum, the signal peptide is cleaved out from the preproinsulin to produce “proinsulin (B-C-A)”. Proinsulin forms disulfide bonds in the endoplasmic reticulum, thereby taking on a three-dimensional structure. Proinsulin is cleaved with a prohormone-converting enzyme PC1/3 at the B-C junction and then cleaved with a converting enzyme PC2 at the C-A junction. Finally, N-terminal two basic amino acid residues of the C-peptide, which remain at the C-terminus of the B-chain when cleaved with PC1/3, are cut out with carboxypeptidase H. In this manner, insulin is produced.
Methods for producing therapeutic insulin have been initially developed using extracts from the pancreas of animals such as bovine and pig. However, human insulin is different in amino acid composition from bovine insulin (at two positions in A chain and one position in B chain) and porcine insulin (at one position in B chain). Therefore, adverse effects (e.g., allergy) are inevitable in the use of bovine or porcine insulin in human bodies. Methods for semi-synthesis of human insulin from porcine insulin have been developed which utilize the transpeptidation reaction with trypsin. However, recombinant insulin produced by genetic recombinant techniques has currently gone mainstream due to its low production cost and good production efficiency.
For the production of recombinant insulin, a number of methods have been developed. For example, the method developed by Eli Lilly Corp. is known, which method comprises expressing A chain and B chain separately using Escherichia coli; and mixing the A chain and the B chain in vitro to form the disulfide bridges, thereby linking them via the disulfide bonds (JP-B- 63-18960). This method, however, is poor in production efficiency. Then, Eli Lilly Corp. has developed an improved method which comprises expressing proinsulin; forming the disulfide bonds in vitro; and then cleaving out the C-peptide from the product with trypsin and carboxypeptidase B, thereby producing insulin (JP-B-1-48278 and Japanese Patent No. 2634176).
Another method was developed by Novo Nordisk Corp., which method comprises expressing miniproinsulin comprising a B chain and an A chain linked via two basic amino acid residues, in yeast; and then treating the miniproinsulin with trypsin in vitro, thereby producing insulin (JP-B-7-121226 and JP-B-8-8871, and Japanese Patent No.2553326). This method has such advantages that the disulfide bonds are formed during the expression and secretion of the miniproinsulin and that the miniproinsulin can be isolated and purified readily because it is secreted into a cultured medium.
Development of new recombinant insulin-production methods has been continued positively. Hoechst Corporation developed a method comprising expressing a new-type insulin derivative or preproinsulin in E. coli; forming the disulfide bonds in vitro; and then treating the product with lysylendopeptidase or clostripain/carboxypeptidase B, thereby producing insulin (JP-A-2-195896, JP-A-2-225498, JP-A-2-233698, JP-A-3-169895, JP-A4-258296, JP-A-6-228191, and JP-A-7-265092). Recently, a method has been developed by BIO-TECHNOLOGY GENERAL CORPORATION, in which a fusion protein comprising superoxide dismutase (SOD) linked with proinsulin is expressed in E. coli to increase both the expression efficiency and the disulfide bond-forming efficiency, and proinsulin is converted into insulin with trypsin and carboxypeptidase B (WO 96/20724). Thus, there are a number of approaches for recombinant insulin production, and further improvement has been made in expression efficiency, disulfide bond-forming efficiency and conversion into insulin.
As the hosts for the production of recombinant proteins, a wide variety of hosts have been used including microorganisms, animals and plants. Among them, microorganisms are most frequently used due to their easy-to-handle property and good applicability for industrial use, and Escherichia coil and yeast are especially known. Recently, an expression system with Bacillus brevis has been known for recombinant proteins (see Japanese Patent No. 2082727; JP-A-62-201583; Yamagata, H. et al., J. Bacteriol. 169:1239-1245, 1987; Juzo Udaka, Nihon Nogei Kagaku-shi 61, 669-676, 1987; Takao, M. et al., Appi. Microbiol. Biotechnol. 30:75-80, 1989; Yamagata, H. et al., Proc. Natl. Acad. Sci. USA 86:3589-3593, 1989).
The object of the present invention is to develop an expression system and a production method for insulin which have a high yield and a production efficiency equal to or better than those of the existing recombinant insulin production systems. That is, the object of the present invention is to develop a novel method for converting an insulin precursor into insulin, an environment where the disulfide bonds necessary for insulin activity can be formed, and an expression system with high yield.